The broad objectives of this proposal are to understand the mechanism of microtubule dynamics in vivo and the relationship between this dynamic activity and important cell biological functions including organization of the cytoplasm, the generation of cell form, cell division and cell locomotion. The conceptual framework, underlying the proposal is that the organization of microtubules and their turnover in cells result from an interplay of cellular components. Two important classes of component are: (1) the centrosome as a nucleating and anchoring structure and (2) cytoplasmic factors that effect the dynamic activity of the minus and plus ends, respectively. Of particular focus in this proposal will be a novel, recently formulated hypothesis for microtubule turnover which we call the minus-end pathway. The specific aims targeted for this grant period are designed to test elements of the minus-end pathway hypothesis and to determine the mechanism by which it operates. The aims are: (1) to determine whether minus end stabilization is dependent on the centrosome in fibroblasts but independent of the centrosome in epithelial cells; (2) to evaluate whether the difference in MT behavior between fibroblasts and epithelial cells is regulated by cell-cell contacts; (3) to determine the status of the MT minus end after its release from the centrosome; (4) to determine the relative contributions of the minus end pathway and plus-end dynamics to the process of MT turnover; (5) to investigate properties of MT treadmilling in vivo and determine the involvement of plus-end factors; and (6) to develop an in vitro MT treadmilling system as an assay for the discovery of minus (and plus) end factors. Our research strategy will employ a combination of kinetic, structural, biochemical, molecular biological and cellular approaches. Novel methods and approaches include the use of centrosome-free cytoplasts as an assay system for microtubule treadmilling; a cell-free system for microtubule release; correlative digital fluorescence imaging and replica electron microscopy; and single microtubule labeling for minus and plus end factors. The results will contribute to an understanding of basic mechanisms of cytoplasmic organization which underlie the maintenance, motility and division of normal as well as malignantly transformed cells.